JPET Fast Forward. Published on January 6, 2009 as DOI:10.1124/jpet.108.147058
JPET #147058 Title page
The smoking cessation medication varenicline attenuates alcohol and nicotine interactions in the rat mesolimbic dopamine system. Mia Ericson, Elin Löf, Rosita Stomberg and Bo Söderpalm.
Addiction Biology Unit, Section for Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg (M.E., E.L., R.S., B.S.) and Beroendekliniken, Sahlgrenska University Hospital (B.S.), Gothenburg, Sweden.
1 Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.
JPET #147058 Running title page Running title: Varenicline attenuates alcohol and nicotine interactions
Correspondence: Elin Löf, Institute of Neuroscience and Physiology, Section of Psychiatry and Neurochemistry, Psychiatry/SU/Sahlgrenska, Blå Stråket 15, SE 413 45 Gothenburg, Sweden. Telephone: +46-31-3424223, Fax: +46-31-828163, E-mail:
[email protected]
Number of text pages: 23 Number of tables: 0 Number of figures: 4 Number of references: 40 Number of words in the abstract: 248 Number of words in the introduction: 531 Number of words in the discussion: 1500
List of abbreviations: nAChRs; nicotinic acetylcholine receptors nAc; nucleus accumbens VTA; ventral tegmental area
Recommended section assignment: Neuropharmacology
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ABSTRACT Varenicline was recently approved as an aid for smoking cessation. Patients treated with varenicline have reported a concomitant reduction of their alcohol consumption. This compound has also been demonstrated to reduce alcohol seeking and consumption in alcohol high-preferring rats. Based on the extensive co-abuse of nicotine and alcohol, the aim of the present paper was to explore whether interactions between varenicline, nicotine and ethanol in the brain reward system could indicate the use of varenicline also for alcohol dependence. Using the in vivo microdialysis method, we investigated the effects of systemic injections of varenicline on the extracellular accumbal dopamine levels in response to a systemic challenge of alcohol, nicotine or the combination of nicotine and alcohol in the experimental rat. Acute systemic co-administration of varenicline and ethanol counteracted each others´ respective enhancing effect on dopamine levels in the nucleus accumbens. However, following 5 days of varenicline pre-treatment, acute combined varenicline and ethanol administration raised dopamine levels to the same extent as either drug alone. Furthermore, after varenicline pretreatment an acute injection of varenicline antagonized the dopamine stimulatory effect of acute nicotine as well as that of systemic co-administration of ethanol and nicotine. In contrast, a pronounced additive dopamine increase was observed when nicotine and ethanol were co-administered in vehicle-pretreated rats. The anti-smoking agent varenicline exhibits properties with respect to its interaction with ethanol and nicotine in the brain reward system that may be beneficial for treating patients with alcohol dependence with (and possibly also without) concomitant nicotine dependence.
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INTRODUCTION Alcoholism is a world-wide problem causing considerable suffering to the individual and enormous costs to society. Three pharmacological compounds are available for the treatment of alcohol dependence (acamprosate, naltrexone, disulfiram). However, in many patients these drugs have low or no efficacy and there is a world-wide increasing need for better pharmacotherapy for this indication. There is a strong correlation between nicotine- and alcohol addiction, where e.g. high alcohol consumption is associated with a high consumption of nicotine, and vice versa (e.g. Zacny, 1990; Daeppen et al., 2000). Extensive research demonstrates that the association between smoking and drinking cannot exclusively be explained by environmental or psychosocial factors. Nicotinic acetylcholine receptors (nAChRs) constitute a common point of action for nicotine and alcohol in their ability to stimulate the mesolimbic dopamine system, an important part of the brain reward system (e.g. Blomqvist et al., 1993); for review, see Soderpalm et al., 2000). In the experimental rat, systemic nicotine or alcohol administration elevates extracellular dopamine levels in the nucleus accumbens (nAc), an effect that requires stimulation of nAChRs in the ventral tegmental area (VTA), where the mesolimbic dopaminergic cell-bodies are located (Blomqvist et al., 1997; Tizabi et al., 2002; Ericson et al., 2003). Moreover, in vitro studies demonstrate that, at behaviorally relevant concentrations, ethanol can act as an allosteric modulator stimulating recombinant human (Cardoso et al., 1999) and rat (Covernton and Connolly, 1997) α4β2* nAChR subtypes. Consequently, nAChRs have been proposed as potential targets for novel selective pharmacological interventions aimed at reducing craving and relapse in alcoholism (Blomqvist et al., 1993; Soderpalm et al., 2000; Balogh et al., 2002; Ericson et al., 2003; Larsson et al., 2004; Jerlhag et al., 2006; Löf et al., 2007). In line with this suggestion, the non-selective nAChR antagonist mecamylamine reduces voluntary alcohol-intake in the
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JPET #147058 experimental rat (Blomqvist et al., 1996; Ericson et al., 1998; Le et al., 2000). Moreover, clinical studies demonstrate that mecamylamine reduces the euphoric and stimulant subjective effects of acute alcohol intoxication in social drinkers, and decreases the subjects´ desire to drink more (Blomqvist et al., 2002; Chi and de Wit, 2003; Young et al., 2005). This nAChR antagonist is, however, associated with substantial side effects, emphasizing the need for new, more selective nAChR modulators for the treatment of alcoholism. Recently, varenicline was registered as a new aid for smoking cessation. It acts as a partial agonist at the α4β2* nAChR subtype, slightly increasing the activity of the mesolimbic dopamine system to alleviate the nicotine withdrawal symptoms, while blocking the reinforcing dopamine-stimulatory properties of nicotine (Coe et al., 2005; Rollema et al., 2007). The clinical efficacy of varenicline (Cahill et al., 2007) is attributed to this dual action of the partial agonist. Several anecdotal reports suggest that patients treated with varenicline to reduce their smoking have reported a concomitant reduction in their alcohol consumption. Moreover, varenicline was recently demonstrated to reduce alcohol seeking and consumption in alcohol high-preferring rats (Steensland et al., 2007). Using the in vivo microdialysis method, the present study investigated the effects of systemic acute or sub-chronic injections of varenicline on the accumbal dopamine response to a systemic challenge of alcohol, nicotine or the combination of nicotine and alcohol in the experimental rat.
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METHODS Animals Male Wistar rats (Beekay, Sweden or Taconic, Denmark) weighing 270-350 g were housed 4 per cage (55x35x20) at constant room temperature (22 °C) and humidity (65%). The animals were kept under regular light-dark conditions (lights on at 7:00 am and off at 7:00 pm) and had free access to “rat standard feed” (Harlan Teklad Europe, UK) and tap water. In all experiments drug naive animals were used. Separate groups of rats were used for each drug/concentration. Animals were allowed to adapt for one week to the novel environment before any experiment was performed. This study was performed in agreement with the Declaration of Helsinki and was approved by the Ethics Committee for Animal Experiments, Göteborg, Sweden (337/06).
Drugs Ethanol (Kemetyl AB, Sweden), varenicline (6,7,8,9-tetrahydro-6,10-methano-6H pyrazino[2,3-h][3]benzazepine tartrate; kindly provided by Pfizer Global Research and Development), and nicotine (nicotine hydrogen tartrate; (S)-3-(1-Methyl-2pyrrolidinyl)pyridine, SIGMA) were all dissolved in 0.9% NaCl and administered systemically. Nicotine doses are expressed as free base.
Microdialysis technique Brain microdialysis experiments were performed in awake and freely moving animals. Rats were anaesthetized by isoflurane, mounted into a stereotaxic instrument (David Kopf Instruments) and put on a heating pad to prevent hypothermia during the surgery. Holes were drilled for the placement of two anchoring screws and an I-shaped dialysis probe custom made in the laboratory. The dialysis probe was lowered into the nAc (A/P: +1.85, M/L: -1.4
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JPET #147058 mm relative to bregma, D/V: -7.8 mm relative to dura; (Paxinos and Watson, 2005)). The dialysis probes were placed in the core-shell borderline region (suggesting that sampling was done in both the core and the shell of the nAc) and the probes and the anchoring screws were fixed to the scull with Harvard cement (DAB Dental AB; Sweden). After surgery, the rats were allowed to recover for two days before the dialysis experiments were initiated. On the experimental day, the sealed inlet and outlet of the probes were cut open and connected to a microperfusion pump (U-864 Syringe Pump, AgnTho’s, Sweden) via a swivel allowing the animal to move around freely. The probe was perfused with Ringer solution at a rate of 2 µl/min and dialysate samples (40 µl) were collected every 20 minutes. The probes were perfused with Ringer solution for one hour in order to obtain a balanced fluid exchange before baseline sampling began. Animals were sacrificed directly after the experiment, brains were removed and probe placements were verified using a vibroslicer (Campden Instruments; Fig.1).
Biochemical assay To analyze the dopamine content of the dialysate samples a high-pressure liquid chromatography system with electrochemical detection was used for the separation and detection of dopamine as previously described (Ericson et al., 2003). To identify the dopamine peak, an external standard was used containing 2.64 fmol/μl of dopamine. When at least three consecutive stable values of dopamine were obtained (± 5%) the first drug was introduced.
Experimental design In the first study we explored the effects of acute application of varenicline on extracellular dopamine levels in the nAc. The animals received an injection of saline or varenicline (0.75,
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JPET #147058 1.5, 3 or 6 mg/kg s.c.) and dialysate samples were collected for an additional 180 minutes. The varenicline doses were chosen based on dopamine turnover in the nAc in one of the original papers describing the effects of varenicline (Rollema et al., 2007). In the second set of animals we explored the possible interaction between varenicline and ethanol. These animals received either varenicline 1.5 mg/kg s.c.+ NaCl i.p., NaCl s.c.+ ethanol 2.5 g/kg i.p., varenicline 1.5 mg/kg s.c.+ ethanol 2.5 g/kg i.p., or NaCl s.c.+ NaCl i.p. and the dopamine levels were monitored for three hours. In the third experiment the animals were dosed with either 0.9% NaCl s.c. or varenicline 1.5 mg/kg s.c. in the afternoon for five consecutive days. This treatment paradigm was used to study the effects of varenicline after repeated administration, a sub-chronic paradigm resembling the one used for aiding human smoking cessation. On the day of the dialysis experiment the animals received a systemic injection of either saline or varenicline (1.5 mg/kg s.c.), 60 minutes before nicotine 0.35 mg/kg s.c.+ saline i.p., saline s.c.+ ethanol 2.5 g/kg i.p. or nicotine 0.35 mg/kg s.c.+ ethanol 2.5 g/kg i.p. after which dopamine levels were monitored for three hours.
Statistics Data were statistically evaluated using a two-way ANOVA with repeated measures (treatment group x time) followed by Fisher’s protected least significant difference test (PLSD) or using a paired t-test. Multiple t-test comparisons were corrected for with Holm´s sequential rejection procedure, a weighted improvement of the Bonferroni correction (Holm, 1979). A probability value (P) less than 0.05 was considered statistically significant. All values are expressed as means ± S.E.M.
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RESULTS
Dose-dependent effect of varenicline. The lowest and the highest doses of varenicline (0.75 mg/kg and 6 mg/kg) did not influence the dopamine output significantly compared to saline administration. However, the intermediate doses (1.5 mg/kg and 3 mg/kg s.c.) elevated and tended to elevate extracellular dopamine levels in the nAc to 55 % (p < 0.05) and 30 % (NS, p = 0.057) of basal levels, respectively (figure 2).
Effect of 1.5 mg/kg varenicline on ethanol-induced dopamine release. We investigated the possible influence of varenicline on ethanol-induced elevation of dopamine levels in the nAc with 1.5 mg/kg varenicline, a dose that produced a robust significant dopamine response. A repeated measures ANOVA demonstrated an effect of drug (figure 3; F[3,26] = 4.858, p < 0.05), a time effect (F[6,156] = 4.379, p < 0.001) and a time x drug interaction (F[6,156] = 2.966, p < 0.001). Separate administration of varenicline or ethanol produced significant elevations of nAc dopamine compared to saline injections (figure 3; Fisher’s PLSD, saline vs. varenicline 1.5 mg/kg p < 0.05, saline vs. ethanol 2.5 g/kg p < 0.05). However, when the two drugs were administered concomitantly, they counteracted each other’s dopamine enhancing properties (figure 3; Fisher’s PLSD, saline vs. varenicline + ethanol p = 0.4922, varenicline + ethanol vs. varenicline p < 0.05, varenicline + ethanol vs. ethanol p < 0.05).
Effect of semi-chronic varenicline on ethanol-induced dopamine release. In the semi-chronic study, animals were pre-treated with either saline or varenicline (1.5 mg/kg) once a day for five consecutive days. On the day of the dialysis experiment, baseline
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JPET #147058 dopamine levels were recorded before drug administration. There were no differences in basal dopamine levels in animals pre-treated with saline (3.18 ± 0.388 fmol/μl) compared to varenicline (2.32 ± 0.298 fmol/μl). Animals that were pretreated with saline received an acute saline injection and animals that were pretreated with varenicline pre-treatment received an acute varenicline injection. One hour later all animals were injected with either nicotine (0.35 mg/kg s.c.), ethanol (2.5 g/kg i.p.) or nicotine plus ethanol and nAc dopamine levels were monitored for another three hours. A repeated measures ANOVA revealed a significant effect of drug (Figure 4; F[5,36] = 2.911, p < 0.05), a time effect (F[12,432] = 20.786, p < 0.0001) and a time x drug interaction (F[60,432] = 1.833, p < 0.001).
Acutely, varenicline produced a significant increase in nAc dopamine levels in varenicline pre-treated animals at time-points 20, 40 and 60 minutes compared to time-point 0 minutes (Figure 4A-B; p < 0.001 in all time-points, paired t-tests corrected for multiple comparisons). In saline pre-treated animals, nicotine produced a significant increase in nAc dopamine levels (figure 4A; p < 0.05, time-point 60 vs. time-points 80 and 100, paired t-tests corrected for multiple comparisons). However, varenicline pre-treatment prevented the nicotine-induced nAc dopamine increase (figure 4A; p ≥ 0.05 time-point 60 minutes vs. all following timepoints, paired t-tests). Ethanol significantly elevated nAc dopamine levels both in vehicle pretreated (figure 4A; p < 0.05, time-point 60 minutes vs. time-point 100 minutes, paired t-tests corrected for multiple comparisons) and in varenicline pre-treated animals (figure 4A; p < 0.05, time-point 60 minutes vs. time-points 80, 100 and 120 minutes, paired t-tests corrected for multiple comparisons). Acute co-administration of nicotine and alcohol produced a significant increase in nAc dopamine in vehicle pre-treated animals (figure 4B; p < 0.05, time-point 60 minutes vs. time-points 80 and 100 minutes, paired t-tests corrected for multiple
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JPET #147058 comparisons), but a significantly lower increase in varenicline pre-treated animals (Fig. 4B; Fisher’s PLSD, p < 0.05).
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DISCUSSION Varenicline was recently approved as an aid for smoking cessation. Based on the extensive coabuse of nicotine and alcohol (Zacny, 1990; Daeppen et al., 2000) and a potential common point of action (nAChRs) for these drugs in the brain reward system (Blomqvist et al., 1993; Soderpalm et al., 2000), the aim of the present paper was to pre-clinically explore the potential benefits of varenicline as a pharmacological treatment of alcohol dependence. Using the in vivo microdialysis method, three sets of experiments measured the effects of subcutaneous injections of varenicline on the accumbal dopamine response to an acute systemic challenge with ethanol or with the combination of nicotine and ethanol in the rat. The first experiment analyzed the impact of different doses of varenicline on extracellular dopamine levels in the nAc. Varenicline is a partial nAChR agonist that binds to
α4β2* nAChRs with greater affinity, but less efficacy, than nicotine (Coe et al., 2005; Mihalak et al., 2006; Rollema et al., 2007). The subsequent effect of varenicline on the mesolimbic dopamine system is, in addition to a blockade of the nicotine-induced dopamine elevation, a slight intrinsic dopamine enhancement that is suggested to alleviate nicotinewithdrawal symptoms (Coe et al., 2005; Rollema et al., 2007). It is plausible that the ability of varenicline to elevate dopamine can provide relief also from withdrawal symptoms and craving related to other drugs of abuse, including alcohol, at least in a certain dose-range. In the present dose-response study, an intermediate dose of 1.5 mg/kg produced the highest dopamine elevation in the nAc, and was consequently selected for the remaining experiments. In line with previous studies (Rollema et al., 2007), the present experiment demonstrated an inverted U-shaped dose-response curve. A possible explanation for this feature could be that, at high doses, varenicline may become an antagonist at nAChRs important for dopamine turnover, alternatively it may start to modulate other nAChR subtypes or non-nAChRs.
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JPET #147058 A second experiment explored whether acutely co-administered varenicline and ethanol interact in the mesolimbic dopamine system with respect to elevation of nAc dopamine overflow. As demonstrated previously, dopamine was increased following acute administration of varenicline alone (Coe et al., 2005; Rollema et al., 2007) and ethanol alone (Blomqvist et al., 1993). Interestingly, dopamine levels were not increased when the same doses of ethanol and varenicline were administered concomitantly, in other words, varenicline appears to block the effect of ethanol and ethanol appears to block the effect of varenicline. Whether there are consequences on the efficacy for the drug in aiding smoking cessation when ethanol is present remains to be established, but no reports have indicated that varenicline has lower efficacy following ethanol consumption in humans. The effect of varenicline on ethanol-induced dopamine overflow in the rat nAc resembles its antagonism of dopamine stimulation by nicotine in the same brain area (Rollema et al., 2007), suggesting that varenicline may be an efficient treatment also for alcohol dependence. This conclusion is supported by behavioral data demonstrating that varenicline reduces ethanol seeking and consumption in ethanol high-preferring rats (Steensland et al., 2007). Moreover, the present results suggest that ethanol and varenicline most likely influence the same nAChR subtypes. Ethanol-induced dopamine elevations in the rat nAc requires stimulation of nAChRs in the VTA (Blomqvist et al., 1997; Tizabi et al., 2002; Ericson et al., 2003). In the rodent VTA, several functional nAChR subtypes have been identified, such as α7*, α4β2* and α6β2β3* (Klink et al., 2001; Wooltorton et al., 2003). Varenicline is mainly considered a selective partial α4β2* nAChR agonist (Coe et al., 2005; Rollema et al., 2007). It is also a full α7* nAChR agonist (Mihalak et al., 2006) and possesses weak, partial agonistic properties at
α3β4*, α6* and α3β2* nAChRs (Mihalak et al., 2006; Rollema et al., 2007). In vitro studies suggest an interaction between ethanol and the α4β2* nAChRs (Aistrup et al., 1999; Cardoso et al., 1999). Moreover, there are several in vivo studies supporting the idea that α4β2*
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JPET #147058 (Owens et al., 2003; Butt et al., 2004) and α7* (Wehner et al., 2004) nAChR are modulated by ethanol, whereas other pharmacological studies in experimental animals appear to exclude the involvement of these nAChR subtypes in several important aspects of alcohol reinforcement. In rodents, the α4β2* nAChR antagonist dihydro-β-erythroidine does not affect alcohol consumption (Le et al., 2000), ethanol-induced locomotor activity (Larsson et al., 2002; Kamens and Phillips, 2007), dopamine overflow (Larsson et al., 2002; Ericson et al., 2003) or conditioned reinforcement to ethanol (Löf et al., 2007). Moreover, the α7* nAChRs antagonist methyllycaconitine citrate had no effect on ethanol-induced dopamine elevations or locomotor activity in rodents (Larsson et al., 2002). Instead, the selective α3β2* and α6* nAChR antagonist α-conotoxinMII blocks the ethanol-induced effects on these parameters (Larsson et al., 2004; Jerlhag et al., 2006; Löf et al., 2007). As mentioned above, varenicline is also a partial agonist at α3β4* nAChRs (Mihalak et al., 2006), although with 800–fold lower binding affinity than for α4β2* nAChRs (Rollema et al., 2007), and blockade of this nAChR subunit composition reduces alcohol (and nicotine) consumption in the rat (Rezvani et al., 1997; Glick et al., 2000; Maisonneuve and Glick, 2003). Taken together, pre-clinical pharmacological studies indicate that other nAChR subtypes than the α4β2* may play a role in the alcohol reinforcement processes. Therefore it cannot be excluded that the ability of varenicline to inhibit the ethanol-induced dopamine elevation in the present study may be mediated via other nAChR subtypes than the α4β2*, despite varenicline´s low potency at these receptors (Mihalak et al., 2006; Rollema et al., 2007). In the last experiment, rats were subchronically pre-treated with varenicline or saline once daily for five consecutive days. On day 6, we investigated the effect of varenicline on the accumbal dopamine response to systemic injections of alcohol, nicotine or the combination of nicotine and alcohol. Varenicline pre-treatment prevented the elevation of dopamine induced
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JPET #147058 by a single nicotine injection. This result is in line with published data demonstrating that acute varenicline abolishes nicotine-induced dopamine overflow in the nAc (see above), but this is the first study illustrating the same effect following subchronic varenicline pretreatment. In contrast, the dopamine elevation following a single acute ethanol injection was unaffected by varenicline pre-treatment, a result that differs from the acute effects of varenicline on ethanol-induced dopamine overflow in the second experiment. Whether the observed dopamine response to varenicline and ethanol represents a failure of varenicline to block the ethanol effect or varenicline´s dopamine elevating effect by itself is, however, difficult to determine, since the dopamine elevations produced by ethanol and varenicline are similar in magnitude and the peak effect of varenicline is probably not reached until approx. 100 minutes following injection (cf. experiment 1). The discrepancies between the effects of acute vs. chronic varenicline administration on drug-induced dopamine elevations may either be explained by alterations induced by the subchronic varenicline regimen or by a different experimental design, i.e. that varenicline was given 60 minutes before ethanol in the last experiment. In any case, the subchronic varenicline administration should be more compatible with the clinical situation, where ethanol would be inflicted upon a relatively constant varenicline exposure. Interestingly, in the last experiment, co-administration of ethanol and nicotine produced an additive effect on the accumbal dopamine levels, whereas co-administration of ethanol and varenicline did not. This result supports the notion that nicotine and ethanol elevate dopamine via different pathways, such as different nAChR subtypes (Larsson et al., 2002; Ericson et al., 2003; Larsson et al., 2004). Moreover, the dopamine stimulatory effect of the combination of nicotine and ethanol was completely abolished by varenicline. If translated to the clinical situation, patients who are treated with varenicline as a smoking cessation aid may not experience an additional dopamine stimulatory effect of alcohol on top of nicotine and/or
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JPET #147058 varenicline. Such a phenomenon could explain why some of these patients report a reduction in their alcohol consumption during treatment with varenicline. The present paper demonstrates different effects of acute varenicline on nAc dopamine response to ethanol in naïve animals compared to varenicline pre-treated animals. The α4β2* nAChR subtype desensitizes following stimulation by physiologically relevant levels of nicotine (Benwell et al., 1995) and is hypothesized to become up-regulated after chronic stimulation (Schwartz and Kellar, 1985; Collins et al., 1994), although data are inconsistent and contradictory. The different effects of acute and subchronic varenicline on the dopamine response to ethanol in the present study may thus be due to compensatory alterations of α4β2* nAChRs provoked by repeated administrations of varenicline. It is also possible that chronic consumption of alcohol and/or nicotine alters the subtype composition of nAChRs towards an increased significance of α4β2* nAChRs also in alcohol reinforcement, thus in favor of the
α4β2* mediated properties of varenicline. In summary, this set of in vivo neurochemical experiments suggests that varenicline reduces neurochemical events associated also with alcohol reinforcement, at least in individuals using nicotine. In addition, to our knowledge this is the first study demonstrating that the antagonist effect of varenicline on nicotine-induced dopamine output in the brain reward system is maintained after sub-chronic treatment.
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ACKNOWLEDGEMENTS
We are grateful to Pfizer Global Research and Development for the generous supply of varenicline.
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JPET #147058 Ericson M, Molander A, Löf E, Engel JA and Söderpalm B (2003) Ethanol elevates accumbal dopamine levels via indirect activation of ventral tegmental nicotinic acetylcholine receptors. Eur J Pharmacol 467:85-93. Glick SD, Maisonneuve IM and Dickinson HA (2000) 18-MC reduces methamphetamine and nicotine self-administration in rats. Neuroreport 11:2013-2015. Holm S (1979) A simple sequentially rejective multiple test procedure. Scandinavian Journal of Statistics 6:65. Jerlhag E, Grotli M, Luthman K, Svensson L and Engel JA (2006) Role of the Subunit Composition of Central Nicotinic Acetylcholine Receptors for the Stimulatory and Dopamine-Enhancing Effects of Ethanol. Alcohol Alcohol 41:486-493. Kamens HM and Phillips TJ (2007) A role for neuronal nicotinic acetylcholine receptors in ethanol-induced stimulation, but not cocaine- or methamphetamine-induced stimulation. Psychopharmacology (Berl). Klink R, de Kerchove d'Exaerde A, Zoli M and Changeux JP (2001) Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. J Neurosci 21:1452-1463. Larsson A, Jerlhag E, Svensson L, Soderpalm B and Engel JA (2004) Is an alpha-conotoxin MII-sensitive mechanism involved in the neurochemical, stimulatory, and rewarding effects of ethanol? Alcohol 34:239-250. Larsson A, Svensson L, Soderpalm B and Engel JA (2002) Role of different nicotinic acetylcholine receptors in mediating behavioral and neurochemical effects of ethanol in mice. Alcohol 28:157-167. Le AD, Corrigall WA, Harding JW, Juzytsch W and Li TK (2000) Involvement of nicotinic receptors in alcohol self-administration. Alcohol Clin Exp Res 24:155-163. Löf E, Olausson P, de Bejczy A, Stomberg R, McIntosh J, Taylor J and Söderpalm B (2007) Nicotinic acetylcholine receptors in the ventral tegmental area mediate the dopamine activating and reinforcing properties of ethanol cues. Psychopharmacology (Berl). 2007 Aug 17; [Epub ahead of print] Maisonneuve IM and Glick SD (2003) Anti-addictive actions of an iboga alkaloid congener: a novel mechanism for a novel treatment. Pharmacol Biochem Behav 75:607-618. Mihalak KB, Carroll FI and Luetje CW (2006) Varenicline Is a Partial Agonist at {alpha}4beta2 and a Full Agonist at {alpha}7 Neuronal Nicotinic Receptors. Mol Pharmacol 70:801-805. Owens JC, Balogh SA, McClure-Begley TD, Butt CM, Labarca C, Lester HA, Picciotto MR, Wehner JM and Collins AC (2003) Alpha 4 beta 2* nicotinic acetylcholine receptors modulate the effects of ethanol and nicotine on the acoustic startle response. Alcohol Clin Exp Res 27:1867-1875. Paxinos S and Watson C (2005) The rat brain in stereotaxic coordinates. Elsevier Academic Press. Rezvani AH, Overstreet DH, Yang Y, Maisonneuve IM, Bandarage UK, Kuehne ME and Glick SD (1997) Attenuation of Alcohol Consumption by a Novel Nontoxic Ibogaine Analogue (18-Methoxycoronaridine) in Alcohol-Preferring Rats. Pharmacology Biochemistry and Behavior 58:615-619. Rollema H, Chambers LK, Coe JW, Glowa J, Hurst RS, Lebel LA, Lu Y, Mansbach RS, Mather RJ, Rovetti CC, Sands SB, Schaeffer E, Schulz DW, Tingley FD, 3rd and Williams KE (2007) Pharmacological profile of the alpha4beta2 nicotinic acetylcholine receptor partial agonist varenicline, an effective smoking cessation aid. Neuropharmacology 52:985-994. Schwartz RD and Kellar KJ (1985) In vivo regulation of [3H]acetylcholine recognition sites in brain by nicotinic cholinergic drugs. J Neurochem 45:427-433.
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JPET #147058 Soderpalm B, Ericson M, Olausson P, Blomqvist O and Engel JA (2000) Nicotinic mechanisms involved in the dopamine activating and reinforcing properties of ethanol. Behav Brain Res 113:85-96. Steensland P, Simms JA, Holgate J, Richards JK and Bartlett SE (2007) Varenicline, an {alpha}4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking. PNAS 104:12518-12523. Tizabi Y, Copeland RL, Jr., Louis VA and Taylor RE (2002) Effects of combined systemic alcohol and central nicotine administration into ventral tegmental area on dopamine release in the nucleus accumbens. Alcohol Clin Exp Res 26:394-399. Wehner JM, Keller JJ, Keller AB, Picciotto MR, Paylor R, Booker TK, Beaudet A, Heinemann SF and Balogh SA (2004) Role of neuronal nicotinic receptors in the effects of nicotine and ethanol on contextual fear conditioning. Neuroscience 129:1124. Wooltorton JR, Pidoplichko VI, Broide RS and Dani JA (2003) Differential desensitization and distribution of nicotinic acetylcholine receptor subtypes in midbrain dopamine areas. J Neurosci 23:3176-3185. Young EM, Mahler S, Chi H and de Wit H (2005) Mecamylamine and ethanol preference in healthy volunteers. Alcohol Clin Exp Res 29:58-65. Zacny JP (1990) Behavioral aspects of alcohol-tobacco interactions. Recent Dev. Alcohol 8:205-219.
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JPET #147058 Footnotes Financial support for this work was obtained from the Swedish Medical Research Council (Diary numbers 2006-4988 and 2006-6385) the Swedish Labor Market Insurance (AFA) support for biomedical alcohol research, the Alcohol Research Council of the Swedish Alcohol Retailing Monopoly, governmental support under the LUA/ALF agreement, Wilhelm and Martina Lundgrens Vetenskapsfond, Fredrik och Ingrid Thurings Stiftelse, Magnus Bergvalls Stiftelse, Gunnar och Märta Bergendahls Stiftelse, Adlerbertska Forskningsfonden, Konrad och Helfrid Johanssons forskningsfond and Sigurd och Elsa Goljes minne.
M.E. and E.L. contributed equally to this work
Reprint requests should be sent to Dr. Elin Löf, Institute of Neuroscience and Physiology, Section of Psychiatry and Neurochemistry, Psychiatry/SU/Sahlgrenska, Blå Stråket 15, SE 413 45 Gothenburg, Sweden. E-mail:
[email protected].
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JPET #147058
Legends for figures
Figure 1. Histology. Location of the microdialysis probes in the nucleus accumbens. The black line indicates the tracks of the microdialysis probes. Numbers beside each plate represent distance from bregma.
Figure 2. Effect of saline or varenicline (0.75, 1.5, 3 or 6 mg/kg s.c.) on extracellular dopamine levels in the nAc as measured by in vivo microdialysis in awake freely moving rats. Drugs were administered as indicated by the arrow. Results are presented as means+SEM, n= 7-8. Statistics: ANOVA with repeated measures followed by Fisher’s PLSD.
Figure 3. Effects of saline, varenicline 1.5 mg/kg s.c., ethanol 2.5 g/kg or the combination of varenicline 1.5 mg/kg s.c. and ethanol 2.5 g/kg i.p. on extracellular dopamine levels in the nAc as measured by in vivo microdialysis in awake, freely moving rats. Drugs were administered as indicated by the arrow. Results are presented as means+SEM, n= 7-9. Statistics: ANOVA with repeated measures over time points 0-60 followed by Fisher’s PLSD
Figure 4. Effects of varenicline or saline pre-treatment (1.5 mg/kg s.c. once a day for five days) on the effects of: A. acute saline or varenicline (1.5 mg/kg s.c.) followed by nicotine (0.35 mg/kg s.c.) or ethanol (2.5 g/kg i.p.) injections. * indicate significant increases in nAc dopamine levels following a varenicline injection compared to time-point 0 minutes (p < 0.001). # indicate significant increases in nAc dopamine levels compared to time-point 60 minutes (p < 0.05) following an acute injection of nicotine or ethanol in saline pre-treated animals and following an acute injection of ethanol in varenicline pre-treated animals.
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JPET #147058 or B. the combination of nicotine and ethanol on dopamine levels in the nAc. * indicate significant increases in nAc dopamine levels following a varenicline injection compared to baseline. # indicate significant increases in nAc dopamine levels compared to time-point 60 minutes (p < 0.05) following acute co-administration of nicotine and ethanol in saline pretreated animals. § indicates a significant difference in nAc dopamine response to the coadministration of ethanol and nicotine in animals pre-treated with saline compared to varenicline (p < 0.05). Drugs were administered as indicated by the arrows. Statistics: ANOVA with repeated measures over time-points -20-220 min followed by Fisher’s PLSD. Data are presented as means + SEM, n= 5-9.
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